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b1-tubulin role in platelet function
Figure 7. A system of competitive polymodification of TUBB1 driven by the expression of TTLL and CCP enzymes is required for platelet production and function. We observe a system by which as induced pluripotent stem cell megacaryocytes (iPSC-MK) mature and express TUBB1, they acquire both polyglutamylated and polyg- lycylated tubulin which co-incides with an increase in the expression of glutamylating and glycylating tubulin tyrosine ligase like (TTL) enzymes and reversing cytosolic carboxypeptidase (CCP) enzymes. A resting platelet is partially polyglutamylated, and on activation the marginal band is further polyglutamylated to drive shape change and spreading through the action of TTLL7. We show that this polymodification spatially affects the position of key motor proteins.
TTLL10 variants maybe associated with moderate to severe bleeding in three unrelated families
Our qRT-PCR screen revealed that a number of TTLL and CCP are upregulated during the process of platelet produc- tion, including the polyglycylase TTLL10. Whole exome sequencing data from patients recruited to the GAPP study identified three unrelated families with rare and novel vari- ants in the TTLL10 gene (Figure 6J; Online Supplementary Figures S12 and S13). Extensive in silico analysis was under- taken on the TTLL10 variants which were predicted as ‘uncertain significance’ using current ACMG guidelines (Online Supplementary Figures S12 and S13).32 Two of the three variants result in frameshifts towards the N-terminus of the protein, preceding the ATP binding region (p.Pro15Argfs*38 [novel] and p.Val249Glyfs*57 [frequency 2.15x10-3]) (Figure 6J). The final family has a missense p.Arg340Trp variant (3.34x10≠5 frequency).
All three families report normal platelet counts, aggrega- tion and secretion, and present with an established history of moderate to severe bleeding, including cutaneous bruis- ing and menorrhagia (Figure 6K). Family A demonstrated a consistently high MPV (normal ranges MPF [fL] (7.83-10.5), while families B and C do not. Interestingly, one of the patients (A 1:1) was re-recruited, and on platelet spreading on fibrinogen coated coverslips we observed a marked increase in platelet area compared to controls when imaged using widefield and single molecule localisation microscopy (SMLM) (Figure 6L).
Discussion
We hypothesized that a system of polymodification (polyglutamylation and polyglycylation) targeting the gluta- mate rich C-terminus of b1-tubulin isoform and analogous to similar PTM demonstrated in cilia and neuronal cells, is a likely mechanism by which the interactions of b1-tubulin with key motors are regulated in both MK and platelets.
We report that mature and proplatelet forming CD42b+ iPSC-MK demonstrate both polyglutamylation and polyg- lycylation (polymodification). We observe a markedly dif-
ferent distribution of these PTM in the resting platelet, where polyglycylation is lost and polyglutamylation is par- tially co-localized to the marginal band. On platelet activa- tion, we observe a marked change in the localization of polyglutamylation specific to the marginal band. In iPSC- MK with a CRISPR KO of TUBB1, we see a complete loss of proplatelet formation and lose the distinct reorganization of polyglutamylated and polyglycylated tubulin around the periphery of MK as seen in WT cells.
MK proplatelet extensions are known to be driven by a system of dynein mediated microtubule sliding, while the marginal band in a resting platelet has been shown to be maintained by the antagonistic movement of dynein and kinesin.13,14,36 Interestingly, polyglutamylation has been reported as a mechanism of altering motor protein proces- sivity, with in vitro assays suggesting that polyglutamylation of b1 tubulin isoforms like TUBB1 and TUBB3 accelerates these motors.28 We show a significant effect of polyglu- tamylation on the spatial localization of dynein and kinesin, supporting in vitro assays which suggest that polyglutamy- lation is an accelerator of motor proteins.
We report two unrelated patient families with rare TUBB1 C-terminal variants (an R359W missense and a L361Afs*19 frameshift linked to macrothrombocytopenia). Interestingly within family A, a second genetic variant in the GFI1B gene, which directly affects the TUBB1 promoter is observed. Individuals within family A positive for the GFI1B variant but WT for TUBB1 have normal platelet counts and a milder increase in MPV and IPF, indicating that the macrothrombocytopenia within the family is possibly linked to the TUBB1 variant, but suggesting a potential additive role for the GFI1B variant which is a focus for future study. Both the clinical phenotype and laboratory investigations points to variable expressivity of the two genetic variants contributing to the overall phenotypes observed.
We go on to show through the expression of these vari- ants in Hek293T cells that each variant results in a dysfunc- tional b1-tubulin protein which is most likely to mirror the effects of a C-terminal truncation.
The system of polymodification evidenced in iPSC-MK
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